STRESS RESPONSES - Cyanobacteria - page 664

transcriptional events. The deduced protein sequence was most similar (70-75%) to the sequence of
ClpB from bacteria and cytosolic form of ClpB from plants. Mutants isolated after gene inactivation
experiments showed no synthesis of ClpB proteins whereas the wild-type produced long (93 kDa)
and short forms (79 kDa) of ClpB after a heat shock.The level of ClpC remained unchanged in the
wild-type but in the ClpB-defective mutant the level of ClpC doubled during heat stress. Taking
photosynthesis as a metabolic indicator, a heat shock (shift-up from 37°C to 55°C for 10 min) caused
a signifi cant reduction in photosynthetic oxygen evolution in wild-type and the mutant. But a pre-
treatment of cells at 50°C for 1.5 h resulted in a signifi cant retention of the photosynthetic effi ciency
in the wild-type. Due to a reduction in the level of ClpB protein in the mutant, it lost the ability to
tolerate the heat stress. These results thus signify the vital nature of the synthesis of ClpB protein
for cell survival at high temperature and for acquiring thermotolerance.
Similarly, cloning and sequencing of ClpB gene from Plectonema boryanum (pClpB) showed it to
consist of two highly conserved ATPase domains characteristic of other ClpB sequences examined.
The accumulation of the transcripts of the small and long forms of pClpB and the respective
proteins due to excess light at low temperature is another signifi cant feature. Phylogenetic analysis
of ClpA , ClpB and ClpC sequences from 27 different organisms (bacteria, yeast mitochondria and
eukaryotes) revealed that the ClpB sequences are well resolved into bacterial and eukaryotic
sequences with the pClpB sequences coming very closer to Synechococcus and other cyanobacterial
ClpB sequences (Celerin et al ., 1998). Clarke and Eriksson (2000) demonstrated that the truncated
form of ClpB (79 kDa protein) has a functional role in conferring thermotolerance in S. elongatus
PCC 7942. Gene inactivation experiments led to the identifi cation of two types of mutants: (i) in
the fi rst type ClpBI-79 was inactivated so that it produced only ClpBI-93 protein and (ii) in the
second type ClpBI-93 was inactivated so that it produced only ClpB1-79. A temperature shift-up
(37°C to 48.5°C) caused a six to seven fold induction of ClpBI-93 protein with a concomitant 2-3
fold increase in ClpBI-79 in the wild-type. However, in the two categories of mutants the levels
of production of the respective ClpBI-93 or ClpBI-79 proteins gave thermal protection to a lower
level than that of the wild-type. The overall data point out that the contribution of the two forms
of ClpB to thermotolerance in wild-type was two-thirds and one-third by ClpBI-93 and ClpBI-79
proteins, respectively. In view of this, they questioned the suggestion that ClpBI-79 might be playing
a regulatory role in the activity of full length ClpBI-93 as has been pointed out in case of E . coli
(Park et al ., 1993). Eriksson et al . (2001) described the existence of a novel form of ClpB (ClpBII) in
S. elongatus PCC 7942 that is not induced by heat or other stresses (light 1000 µmol photons m -2 s -1 ;
cold 25°C; salt 150 mM NaCl or H 2 O 2 0.5 M). Though a constitutive protein, ClpBII was unable to
complement ClpBI in its function for protecting the cells against heat stress as evidenced by the gene
inactivation experiments. Thermotolerance assays (at 54°C for 15 min after a fi rst pre-treatment at
48.5°C for 1.5 h) showed that the wild-type developed signifi cant thermotolerance over ClpBI- and
ClpBII-defi cient mutants. The ClpBII gene is a single copy ORF (with 2,685 bp) without the typical -10
mer or -35 mer promoter sequences characteristic of E . coli genes. It possesses two ATP-binding domains
(ATP1 and ATP2) that are separated by a spacer of 129 amino acids. Unlike ClpBI that produces a full
length (93 kDa) and a truncated form of the protein (79kDa), ClpBII produced a single form of ClpBII
(97 kDa) polypeptide that corresponded to the predicted size of ClpBII.
The multigene family of ClpP from S . elongatus PCC 7942 has been characterized. The three ClpP
genes appeared to be different in their organization. ClpPI gene is monocistronic in contrast to ClpPII
and ClpPIII genes that are part of bicistronic operons with ClpX and ClpR , respectively. Though not
inducible by a heat (50°C for 2 h) or cold (25°C for 6 h) shock or high light (1000 µmol photons m -2
s -1 ) or oxidation (H 2 O 2 0.5 M), ClpPII , ClpPIII , ClpX and ClpR seemed to be essential for cell viability.